Chemokine receptors and their ligands direct the trafficking of cells in normal tissue homeostasis and in disease, influencing cell motility, invasiveness and survival [1]. In inflammation and in cancer, chemokines in the diseased tissues contribute to the rolling, tethering and invasion of leucocytes from the blood vessels through the endothelial cell basement membrane and into the parenchyma [2].
CCR4 is one of 18 known chemokine receptors. Chemokine receptors are generally expressed on immune cells and in the tumour microenvironment a number of receptors and their ligands are present in the immune cell infiltrate.
In many cancers, malignant cells also express certain chemokine receptors, receptors that are not usually found on their normal counterparts. Metastatic cancer cells are thought to gain characteristics of chemokine receptor-expressing leucocytes, using chemokines to aid their migration to, and survival at, sites distant to the original tumour [3, 4, 5]. Inappropriate presence on cancer cells of chemokine receptors that usually have a highly restricted pattern of expression further supports the hypothesis that specific chemokine receptors may help cells spread to, and/or survive in, different metastatic sites [8]. In carcinomas, melanomas and haematological malignancies, expression of chemokine receptors, especially CXCR4 and CCR7, on malignant cells in advanced disease, correlates with increased lymph node metastasis, greater dissemination of disease, lower disease-free survival and/or overall survival [6,7,8]. CXCR5 is normally restricted to B cells and some T cell subtypes, but is also expressed by pancreatic cancer cells where it is implicated in the establishment of liver metastases; the liver being a site of production of the CXCR5 ligand, CXCL13 [9]. Melanoma cells that have metastasised to the intestine express CCR9 [10]. In homeostasis, the CCR9 ligand CCL25 recruits rare T cell subsets to the intestine. In pancreatic cancer, expression of CCR6 has been observed [38][39]. CCR6 expression has also been reported in human renal carcinoma, together with CCR3 and CXCR2 [40].
This demonstrates that a few chemokine receptors are known to be upregulated in tumour epithelial cells in late stage carcinogenesis, including CXCR4, and are thought to play a role in invasion and metastasis. In contrast, CCR4 has only previously been reported to be upregulated in some blood cancers, particularly T cell lymphomas.
As described above, CXCR4 is commonly found on malignant cells in many advanced human cancers. In addition, Woerner et al found that CXCR4 was also present in the early stages of disease in glioblastoma [20]. However, using a phospho-specific anti-CXCR4 antibody, they found that in the less malignant Grade 1 lesions, the level of receptor activation was much lower.
Although it is generally reported that malignant cell chemokine receptor expression is associated with advanced disease, there are also a few other reports in the art of expression of a chemokine receptor on malignant cells at early and pre-invasive stages of cancer, but all of these concern CXCR4. In a large tissue array study (over 2000 samples) of breast cancer, cytoplasmic/membrane expression of CXCR4 expression was reported in 67% of ductal carcinomas in situ, DCIS [21]. This was confirmed in a study from Schmid et al who showed that both CXCR4 and its ligand CXCL12 were expressed in DCIS [22]. The inventors have also found CXCR4 on the epithelial cells of borderline non-invasive ovarian cancer tumours (Kulbe et al., manuscript in preparation) and this has also been reported by Pils et al [23].
No data on expression of other chemokine receptors on epithelial cells in early cancers is available, but there is evidence that oncogenic pathways can induce chemokine receptor expression on epithelial cells. The RET/PTC1 oncogene is necessary and sufficient for malignant transformation of primary thyrocytes [24]. This oncogene induces a pro-inflammatory programme in the thyrocytes that includes induction of functional CXCR4. Alveolar rhabdomyosarcoma is a highly aggressive tumour characterised by recurrent PAX3 and PAX7-FKHR gene fusions. Transfer of PAX3-FKHR into embryonal rhabdomyosarcoma cells also activates CXCR4 expression [Libura, 2002 #9346].
In all these studies a conclusion is that acquisition of certain chemokine receptors by malignant cells appears to be, a relatively late event in malignant progression, and in the case of CCR4, expression has not been reported at any stage of solid tumour development
CCR4 expression is generally restricted to the immune system, and is known as a marker of Th2 and regulatory T cells. In the tumour environment, these cells act to suppress cytotoxic T cells and dendritic cell maturation, hence suppressing anti-tumour immune responses. In addition, CCR4 has been shown to be expressed in haematological malignancies, including by a high proportion of adult T cell lymphomas (ATL), and was a significant prognostic factor associated with metastasis to skin [35], [41]. As such, CCR4 is of interest as a therapeutic target in ATL [37], [42]. CCR4 expression by adult T cell leukaemia is associated with skin metastases; its ligands CCL17 and CCL22 are produced by both malignant cells and the skin tumor microenviroment [36]. Ishida et al have developed an anti-CCR4 monoclonal antibody therapeutic for the treatment of adult T cell lymphoma that induces ADCC activity against the tumor cells and may also act on immunosuppressive malignant Treg cells found in this disease [37].
The only report in the academic literature of a CCR4 positive solid tumor cell line is the human lung cancer cell line SBC-5 [34]. These cells migrated towards CCL22 gradients and in bone metastatic SBC-5 xenografts there was close co-localisation of osteoclasts expressing CCL22 and SBC-5 cells expressing CCR4. There are no reports of CCR4 expression in primary human tumour cells.
WO05106471 (BAYER HEALTHCARE AG) discloses screening methods for agents of potential use in treating a wide range of diseases; specifically consisting of cardiovascular disorders, gastrointestinal and liver diseases, inflammatory diseases, metabolic diseases, haematological disorders, cancer disorders, neurological disorders, respiratory diseases and reproduction disorders in a mammal. The screening method determines the degree of binding or otherwise of candidate agents to CCR4. There is also a description of the screening of a wide range of human cells and tissues for their expression level of CCR4 relative to housekeeping gene expression. The cells and tissues were obtained from disparate sources and just an isolated few were cancerous cells/tissues; e.g. thyroid, ileum, HeLa, Jurkat, lung and breast cancer cells. The results for the relative expression of CCR4 show no distinguishable pattern associated with any particular disease. Indeed amongst tumour cells tested, e.g. thyroid and ileum, there were low levels of relative expression of CCR4 and other non-tumour cells showed higher levels of relative expression of CCR4.
WO9623068 (GLAXO GROUP LIMITED) discloses a chemokine receptor able to bind to Monocyte Chemotactic Protein-1 (MCP-1/CCL2), Macrophage Inflammatory Protein 1α (MIP 1α/CCL3) and/or ‘RANTES’ (Regulated upon Activation, Normal T-cell Expressed, and Secreted/CCL5). A nucleotide and an amino-acid sequence for CCR4 are disclosed (CC-CKR-4/K5.5. K5.5 and CC-CKR-4 are alternative names for CCR4.) The expression of CCR4 is discovered in a relatively limited range of normal human tissues and in a range of T-cell samples. There is also general disclosure of screening assays for agents capable of activating T-lymphocytes or blocking binding of ligands MCP-1, MIP-1α and/or RANTES to the chemokine receptor. There is some suggestion that active agents obtained via screening may be useful in the treatment of allergies, for example.
WO0041724A1 (LELAND STANFORD/LEUKOSITE) proposes the modulation of systemic memory T cell trafficking by administration of CCR4 modulating agents. This is intended as a treatment for inflammatory skin disease. Substances capable of modulating CCR4 binding to its ligands are used in in vitro tests to show how T-cell migration is affected.
Antibodies reactive against CCR4 are known. WO0164754 (Kyowa Hakko Kogyo) discloses a recombinant antibody or fragment thereof allegedly reactive specifically with the extracellular domain of CCR4. Also disclosed is a polypeptide sequence of such an antibody. There is also disclosed an antibody which reacts with a CCR4 positive cell and is cytotoxic or causes antibody-dependent cell-mediated cytotoxicity (ADCC.) These antibodies are proposed for the use in the treatment of Th2-mediated immune diseases or blood cancer, specifically leukaemia.
WO05035582 (Kyowa Hakko Kogyo) discloses an antibody capable of specifically binding CCR4 and also discloses a CCR4 antibody which has a complex N-linked glycosylation in the Fc region. Also disclosed are antibodies to the extracellular domains of CCR4.
WO03018635 (Kyowa Hakko Kogyo) discloses ‘Human CDR-grafted antibodies and fragments’. A specific CDR (complementarity determining region) which binds specifically to CCR4 is disclosed. The antibodies are proposed for use in the diagnosis or treatment of Th2-mediated immune diseases or cancers such as blood cancers.
WO05053741 (Kyowa Hakko Kogyo) discloses a medicament comprising a recombinant antibody, which specifically binds CCR4, in combination with at least one other agent. The antibody is proposed for the treatment of tumours, specifically haematopoietic organ tumours.
WO0042074 (MILLENIUM PHARMACEUTICALS) discloses antibodies to CCR4 and antibodies that can compete with their binding. No specific diagnostic applications are disclosed. Therapy of inflammatory disorders is proposed.
Also known in the art are a variety of small molecules that bind to the CCR4 receptor.
WO4007472 (ONO PHARMACEUTICAL CO.) discloses a small molecule tricyclic compound with anti-CCR4 activity.
WO05023771 (ONO PHARMACEUTICAL CO.) discloses small molecule nitrogen-containing heterocyclic compounds with anti-CCR4 activity.
WO02094264 (TULARIK INC.) discloses specific compounds with CCR4 inhibitory activities.
WO0230358 (TULARIK/CHEMOCENTRYX) discloses various CCR4-binding compounds and uses for treatment of various diseases, but not including cancer.
WO0230357 (CHEMOCENTRYX) discloses compounds that are antagonists of
CCR4. This application describes uses for the treatment of inflammatory diseases and conditions.
WO051236976 (ASTELLAS PHARMA INC.) discloses quinazoline derivatives as CCR4 regulators.
WO05085212 (YAMANOUCHI PHARMACEUTICAL CO., LTD.) discloses pyrimidine derivatives as CCR4 modulators.
WO05082865 (YAMANOUCHI PHARMACEUTICAL CO., LTD.) discloses fused bicyclic pyrimidine derivatives as CCR4 function-controlling agents.
WO04108717 (ASTRAZENECA AB) discloses sulphonamide compounds that modulate chemokine (specifically CCR4) receptor activity.
EP1633729 (ASTRAZENECA AB) discloses sulphonamide compounds that modulate chemokine (specifically CCR4) receptor activity.
WO03014153 (TOPIGEN PHARMACEUTIQUE INC.) discloses another technology in the art, a method of modulating viral infection of a cell by modulating the interaction between chemokine receptors (including CCR4) and a virus.
WO2004/045526 (Morehouse School of Medicine) discloses antibodies to particular chemokines and chemokine receptors and their use in inhibiting the growth and metastasis of cancer cells. Antibodies were raised against the particular chemokine receptors and their ligands, which does not include CCR4. Also described are methods of testing for over-expression of particular chemokines in a tumour and the suggestion that such tumours can be treated by administering antibodies against the particular over-expressed chemokine or chemokine receptor.
WO99/15666 (Icos Corporation) discloses nucleotide sequences and polypeptide sequences of a macrophage-derived C-C chemokine designated ‘Macrophage Derived Chemokine’ (MDC). MDC appears synonymous with CCL22. TARC appears synonymous with CCL17. Methods for the recombinant or synthetic production of MDC protein or polypeptide fragments are described. Also disclosed are antibodies reactive with MDC as well as assays for identifying modulators of MDC and TARC chemokine activity.
Cervical cancer is the second most common type of cancer in women worldwide. Symptoms are often absent until the cancer is at a late stage and hence cervical cancer has been the subject of an intense population screening program using the Pap smear, which can detect pre-malignant changes by histopathology. Although an abnormal Pap smear indicates possible cervical neoplasia, it is insufficient for diagnosis, which is subsequently carried out by biopsy and additional invasive procedures (‘colposcopy’). In total, 24,000 women are referred in the UK each year with abnormal Pap smears. The Pap smear has only 70% sensitivity, hence a significant proportion of women with cervical cancer or pre-invasive lesions remain undiagnosed. Therefore, more accurate screening methods are required to i) allow screening to be more automated and less subjective ii) to improve the sensitivity of screening.
HPV (Human Papilloma Virus) infection is found in the majority of invasive cervical carcinomas, one strategy is to screen for the presence of HPV markers such as E6 and E7 in concert with the Pap smear. However, due to the high level of HPV infection in the sexually active population (up to 80% infection history), this also results in the identification of a large number of false positives and makes the accuracy of the test dependent on HPV prevalence. As such, identifying new biomarkers for cervical cancer remains an area of active interest.
Furthermore new or alternative biomarkers are required for other forms of cancer including, but not limited to, the following cancer types: bronchial, nasopharyngeal, laryngeal, small cell and non-small cell lung, skin (e.g. melanoma or basal cell carcinoma), brain, pancreatic, neck, lung, kidney, liver, breast, colon, bladder, oesophagus, stomach, cervical, ovarian, germ cell and prostate. A biomarker characteristic of one cancer type may be shared with other cancer types thus the use of a biomarker may extend beyond the original cancer type it was found to be associated with.
There is a need for improved biomarkers for a range of cancers which allow for stratification of patients in need of anti-cancer treatment.
The stage of a cancer is a descriptor (usually numbers Ito IV) of how much the cancer has spread. The stage often takes into account the size of a tumour, how deep it has penetrated, whether it has invaded adjacent organs, if and how many lymph nodes it has metastasized to, and whether it has spread to distant organs. Staging of cancer is important because the stage at diagnosis is the most powerful predictor of survival, and treatments are often changed based on the stage
Correct staging is critical because treatment is directly related to disease stage. Thus, incorrect staging would lead to improper treatment, and material diminution of patient survivability. Correct staging, however, can be difficult to achieve. Pathologic staging, where a pathologist examines sections of tissue, can be particularly problematic for two specific reasons: visual discretion and random sampling of tissue. “Visual discretion” means being able to identify single cancerous cells intermixed with healthy cells on a slide. Oversight of one cell can mean mis-staging and lead to serious, unexpected spread of cancer. “Random sampling” refers to the fact that samples are chosen at random from patients' lymph nodes and are examined. If cancerous cells present in the lymph node happen not to be present in the slices of tissue viewed, incorrect staging and improper treatment can result.
There is an ongoing need for new treatments against cancer, whether these involve improved ways of administering existing anti-cancer agents, or whether these involve identifying, testing and verifying effective new anti-cancer agents. There is also an ongoing need for improved methods of monitoring the efficacy of existing and any new anti-cancer agents in the course of a given treatment regime. Improved methods of generating data of predictive value are needed. The dosage and frequency of treatments using anti-cancer agents is an important factor. Also, the timing of the start of an anti-cancer treatment relative to the stage of progression of a cancer, or relative to a patient group, are important factors. Improved methods of monitoring are required in order to seek optimal treatments for patients, whether as individuals or classified into groups by virtue of genetic, phenotypic or other characteristics.
An example of the prognostic function of a biomarker in the choice of treatment for a patient is the use of the anti-cancer drug Herceptin (Trastumuzab). The HER2/neu gene is a proto-oncogene located at the long arm of human chromosome 17(17q11.2-q12) and amplification of HER2/neu occurs in 25-30% of early-stage breast cancers. In cancer the growth promoting signals from HER2/neu are constitutively transmitted, promoting invasion, survival and angiogenesis of cells. Furthermore overexpression can also confer therapeutic resistance to cancer therapies. Herceptin (Trastumuzab) is a humanised monoclonal antibody which binds to the extracellular segment of the receptor HER2/neu, (also known as ErbB-2) and is only effective in treating breast cancer where the HER2/neu receptor is overexpressed. Because of its prognostic role as well as its ability to predict a patient's response to Herceptin breast tumors are routinely checked for overexpression of HER2/neu by a variety of techniques including immunohistochemistry (IHC) Chromogenic and fluorescence in situ hybridisation (CISH and FISH respectively).
There also exists the need for more accurate and reliable methods of diagnosing/staging of cancers and a need for new methods for screening anti-cancer agents.